11/1 Enzymes Part Ii (Not Finished)

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ATCase general info

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Aspartate Transcarbamoylase

In E.coli

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ATCase subunit arrangement

Location of active site

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6 regulatory subunits
6 catalytic subunits

active sites are formed from residues from two catalytic subunits at the interface

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ATCase Active site rxn

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Carbamoyl phosphate binds via electrostatic and h bond.

Asp binds, its amino attacks carbonyl carbon

Tetrahedral transition state

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ATCase inhibitors

What's its use?

How does it affect ATCase?

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PALA as competitive inhibitor artificially. CTP normally.

Helped solve the structure.

Can bind up to 6, makes large conformational changes.
CTP: stabilizes the T state (inactive)

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What all regulates ATCase?

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its substrate
CTP
PALA

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The actually sigmoidal curve is calculated how?

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Average of the pure R-state curve and pure T-state curve

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What allostery model does ATCase follow?

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concerted mechanism (all or nothing)

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Isozymes. Similarities and Differences

Why do we bother with this?

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Catalyze the same reaction

Differ in sequence, regulation, substrate affinities, kinetics.

Can fine-tune processing of particular substrates/products

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Lactate Dehydrogenase forms

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H form in heart. M form in skeletal muscle
H4 M4 tetramers
H has higher affinity.

Can make heterotetramers to allow for different affinities.

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Covalent Modifications (not considered allostery)

Two types with examples

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Reversible (only because we have enzymes to reverse it)
phosphorylation, acetylation
(kinases and phosphatases put P on/off)

Irreversible
attachment of lipidd group that localizes the protein in the membrane

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What residues get phosphorylated?
By what?
With what?

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Ser, Thr, Tyr
Protein kinases
Phosphoryl group from the gamma phosphoryl group of ATP

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What removes phosphoryl groups?

Using what?

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Protein phosphatase

Water

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Why are phosphoryl groups the ones used to regulate?

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Adds 2 negative charges (change electrostatic)
Can form up to 3 h bonds (changes binding)
Free energy is large (change equilibria as E is stored in protein)
Can add and take off quickly or slowly
Can be highly amplified (kinase can act on hundreds of targets)
ATP is energy currency, integrates energy status in rxns

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What terms are used to describe kinase specificity?

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multifunctional - phos many different targets
dedicated - phos a single protein or family of closely related proteins

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PKA aka

Sequence it acts on

Structure style

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protein kinase A or cAMP-dependent protein kinase

RRXSZ or RRXTZ X: small Z : large hydrophobe

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PKA structure style

In the presence of cAMP...

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2 large regulatory subunits R and two smaller catalytic subunits C

.. the protein dissociates into R2 and two active C units

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How does the regulatory subunit work?

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Has sequence RRGAI (a pseudosubstrate sequence)

Binding cAMP to R subunit allosterically moves pseudosubstrate sequence out of catalytic sites

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Using proteolytic cleavage: idea

What is the thing you activate?

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Activate by cleaving

Zymogens

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Some examples of zymogens

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Digestive enzymes: localization
blood clotting: cascade activations
Hormones: insulin
Collagen/collagenase
Caspases: proteolytic enzymes for apoptosis

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Activation of blood clotting

Two pathways

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A series of zymogen activations: cascade
Very small amounts of initial factors make cascade which makes large amplification

Intrinsic: rupture of blood vessels
Extrinsic: tissues releases clotting substances in response to trauma

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What actually composes a clot?

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Fibrinogen has three globular units and rod of ahelical coiled coils

Thrombin cleaves four bonds in central glob to release A peptide from each alpha chain, B from Beta chains

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How is thrombin regulated?

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Synthesized as prothrombin.

After cleavage thrombin has serine protease active site

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What is the essential substance for thrombin?

What if you don't have any?

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Vitamin K

Anticoagulatns are vit K antagonists. If you don't have vit K you get abnormal prothrombin

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What is wrong with abnormal prothrombin?

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Can't bind Ca2+ at the N-terminal as it usually does

There are gamma-Carboxyglutamate normally that binds the Ca2+

Ca2+ anchors prothrombin in phospholipid membranes following injury to bring zymogen into close proximitiy to Factor X_a_ and Factor V

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Hemophilia. How does it work?

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X-linked recessive

Factor VIII (antihemophilic factor) stimulates Factor X -> Xa by Factor IXa (serine protease)